Substituted nitroimidazolylthiadiazoles and oxadiazoles as antiprotozoal agents

ABSTRACT

WHEREIN R IS SELECTED FROM THE GROUP CONSISTING OF LOWER ALKYL, HYDROXY LOWER ALKYL, LOWER ALKANNOYLOXY LOWER ALKYL AND BENZYL; X IS SELECTED FROM THE GROUP CONSISTING OF OXYGEN AND SULFUR; AND   -N(-R1)-R2   TAKEN TOGETHER IS SELECTED FROM THE GROUP CONSISTING OF 1IMIDAZOLYL AND 1-PYRROLIDINYL, OR PHYSIOLOGICALLY ACCEPTABLE SALTS THEREOF. 1. A METHOD OF INHIBITING THE GROWTH OFF PROTOZOA IN WARM-BLOODED ANIMALS WHICH COMPRISES ADMINISTERING ORALLY OR PARENTERALLY TO SAID ANIMALS AN ANTIPROTOZOALLY EFFECTIVE AMOUNT OF A NITROAMIDAZOLE OF THE FORMULA:   1-R,2-((-X-C(-N(-R)-R2)=N-N=)&gt;C-),5-(O2N-)-IMIDAZOLE

United States Patent O 3,842,174 SUBSTITUTED NITROIMIDAZOLYLTHIADI- AZOLES AND OXADIAZOLES AS ANTI- PROTOZOAL AGENTS Gerald Berkelhammer, Princeton, and Com Asato, Titusville, N.J., assignors to American Cyanamid Company, Stamford, Conn.

No Drawing. Application Jan. 12, 1972, Ser. No. 217,709,

now Patent No. 3,740,434, which is a division of application Ser. No. 30,944, Apr. 22, 1970, now Patent No. 3,666,860, which is a continuation-in-part of abandoned application Ser. No. 814,160, Apr. 7, 1969, which is a continuation of application Ser. No. 659,596, Aug. 10, 1967, now Patent No. 3,452,035, which in turn is a continuation-in-part of abandoned application Ser. No. 604,158, Dec. 23, 1966. Divided and this application Mar. 5, 1973, Ser. No. 338,129

Int. Cl. A61k 27/00 US. Cl. 424-270 4 Claims ABSTRACT OF THE DISCLOSURE The use of substituted nitroimidazolyl-thiadiazoles and oxadiazoles are described along with methods of administration of the same. These compounds have been found useful in controlling protozoa and therefore, are antiprotozoal agents.

This is a division of application Ser. No. 217,709 filed Jan. 12, 1972, now Pat. No. 3,740,434 which is a divisional application of our copending application Ser. No. 30,944, filed Apr. 22, 1970, now Pat. No. 3,666,806, which is a continuation-in-part of our application Ser. No. 814,160, filed Apr. 7, 1969, now abandoned, which is a continuationof application Ser. No. 659,596, filed Aug. 10, 1967, now US. Pat. 3,452,035, which in turn is a continuation-inpart of our application Ser. No. 604,158, filed Dec. 23, 1966, now abandoned.

BACKGROUND OF THE INVENTION The present invention describes the use and method of administration of compounds which are both highly effective at relatively low concentrations against a broad spectrum of protozoa, and additionally provide a relatively satisfactory margin of safety. The compositions containing substituted imidazolyl compounds of the present invention are at least 5 to times more active than certain distantly related imidazolyl compounds of the prior art, providing effectiveness at relatively low concentrations, as well as satisfactory margins of safety.

Imidazolyl compounds in which the heterocyclic rings are joined by a methyleneamino bridge have been prepared and found to have some antiprotozoal activity; however, such compounds have not been entirely satisfactory for these purposes. The concentration at which such compounds are active is generally much higher than the desirable level; therefore, said compounds do not provide a satisfactory margin of safety. Illustrative of said compounds which have been prepared and found to have such activity are, for example, 3 [(1 methyl 5 nitro-2- imidazolyl methylene)amino]-2-oxazolidinone and 1-[(1- methyl-S-nitro 2 imidazolylmethylene) amino]-2-imidazolidinone.

SUMMARY OF THE INVENTION The subject matter of the present invention relates to compositions containing as the active component l-substituted 5 nitro 2 imidazolyl compounds and an edible carrier. More particularly, the invention relates to 3,842,174- Patented Oct. 15, 1974 "ice compositions containing compounds having the following general formula:

Wang.

taken together is selected from the group consisting of N=CHN (lower alky1) l-imidazolyl, piperazinyl, N- lower alkylpiperazinyl, N-hydroxy lower alkylpiperazinyl, N-('N',N'-dilower-all(ylamino)lower al kylpiperazinyl, N- pyridylpiperazinyl, N-thiazolylpiperazinyl, N -lower alkoxycarbonyl piperazinyl, piperidino, hydroxypiperidino, l-pyrrolidinyl, morpholino, dilower alkylamino lower alkylpiperidino; and physiologically acceptable salts thereof. The term lower alkyl as employed in the instant specification and claims is intended to include either straight or branched chain having from 1 to 4 carbon atoms. The term halogen is intended to include chlorine, bromine, iodine and fluorine. Physiologically acceptable salts can be, for example, hydrochloride, sulfate, succinate, maleate, mucate, phosphate and the like. The invention relates to a method of inhibiting the growth of protozoa in a warmblooded animal host which comprises administering to the animal therapeutically effective quantities of the above compounds and thereby alleviating diseases, as well as composition of matter comprising said compounds and an edible carrier.

The compounds of the present invention are effective against infections caused by Trichomonas vaginalis, Eimeria tenella, H istomonas meleagridis, Eimeria maxima, Eimerz'a brunetti, Eimeria necatrz'x, Trypanoaoma cruzi, Trypanoaoma equiperdum and ameba.

Preparation of substituted imidazolyls of the present invention In many instances the substituted imidazolyl compounds comprising the active component of the present compositions are prepared by oxidative cyclization of various thicsemicarbazones and semicarbazones of l-substituted-S- nitro 2 imidazolecarboxaldehyde. Suitable oxidizing agents for such cyclization of the thiosemicarbazone to aminothiadiazoles include a wide variety of ferric salts such as ferric ammonium sulfate, ferric chloride, ferric nitrate, ferric acetate, sodium ferricyanide, sodium ferric oxalate, potassium ferric sulfate, and the like. The cyclizations of the semicarbazones to aminooxadiazoles are generally carried out with such agents as sodium hypobromite, sodium hypoiodite, and bromine with sodium acetate. The reactions are generally carried out at an elevated temperature between 50 and C., depending on the particular moiety being prepared.

A number of the 2-(2-substituted amino-S-thiadiazolyD- 1substituted-S-nitroimidazoles can be prepared by the re action of 2-(2-halo-5-thiadiazolyl) l substituted-S-nitroimidazoles with the appropriate primary or secondary amines in an organic solvent, either in the presence of excess amine or with the use of other acid acceptors, as for example, aqueous sodium bicarbonate solution, usually at between 25 and 125 C.

in the preparation of active components having the above general formula wherein R represents a hydroxy lower alkyl group, it is often practical to first synthesize the corresponding ester, as for example, a compound of the formula:

N lII--N H O2NALNJIKXJ&IM

wherein R is lower alkanoyloxy lower alkyl and X and R are as defined hereinabove, and then treat the thus formed ester with a strong mineral acid followed by pH adjustment to above about pH 7, thereby yielding the desired product. In many cases, it is found that the conditions used for cyclization 1-(2-lower alkanoyloxy lower alkyl)- -nitro-2-imidazole carboxaldehyde thiosemicarbazones are sufiiciently acidic to give rise directly to the 2-(2- amino-S-thiadiazolyl)-1-(hydroxy lower alkyl)-5-nitroimidazoles.

Where it is desired to prepare the 2-(2-formamido-5- thiadiazolyl) 1-substituted-nitroimidazole, or the 2-(2- acyl amino or 2 haloacylamino 5 thiadiazolyl) 1- substituted-S-nitro-imidazole, it is practical to first synthesize the 2-(2-amino-5-thiadiazolyl) l-substituted-S nitroimidazole, and then treat the product with formic acid, in the case of the preparation of the 2-(2-formamido-S-thiadiazolyl) 1-substituted-S-nitroimidazol or with an anhydride of the formula (lower alkanoy) or (halo lower alkanoyl) or with the appropriate acid chloride in the instance of the preparation of the 2-(2-acylamino or 2-haloacyl-amino-S-thiadiazolyl)-l-substituted- 5 nitroimidazole. Similar reactions can be carried out to give the corresponding oxadiazolyl compounds. These reactions are usually carried out at an elevated temperature, particularly at temperatures between 50 and 150C.

The 2-(2-amino-5-thiadiazolyl) l-lower alkanoyloxy lower alkyl-S-nitroimidazoles and the corresponding oxadiazolyl compounds, active components of the present compositions, can be made by esterifying the 2-(2-amino- S-thiadiazolyl or 5-oxadiazolyl) l-hydroxy lower alkyl- 5-nitroimidazoles by heating with an aliphatic acid in the presence of a mineral acid catalyst. If, instead, an aliphatic acid anhydride is employed, the products are 2-(2-lower alkanoylamino 5 thiadiazolyl or 5-oxadiazolyl)-1- lower alkanoyl lower alkyl-S-nitroimidazoles. In the latter case, a catalyst is usually not necessary. In both instances, reaction temperature of from 50 to 150 C. are frequently employed.

For the preparation of N,N-dilower alkyl-N'-[5-(1-substituted 5-nitro 2-imidazolyl)thiadiazol 2-y1]formamidines, products of the reaction of N,N-dilower alkyl formamides with phosgene, phosphorus oxychloride, or thionyl chloride are reacted with 2-(2-amino-5-thiadiazolyl)-1-substituted S-nitroimidazoles in an organic solvent, as for example, in an excess of the N,N-dilower alkyl formamide, usually at the ambient temperature.

The active components of the compositions of the instant invention are highly effective in controlling protozoal infections in a warm-blooded animal host; said active components may be administered to the warm-blooded animals in admixture with their feed or drinking water. Furthermore, the compositions may be administered in the form of tablets, pills, capsules or the like, or parenterally by injection either intramuscularly or subcutaneously. The concentration employed in feed or water may be in the range of from 5 to 1,000 parts per million, preferably to 500 parts per million; the most preferred concentration being about 50 to 200 parts per million. When administered parenterally, generally about 2.0 to 30 mg./kg. and preferably 5 to 20 mg./kg. of body weight provides effective disease control. The above compositions have demonstrated effectiveness against Salmonella infections when the active component was administered in as little as 0.025% concentration in the diet of chicks and mice. Furthermore, the active components of the present compositions have shown significant control of Trichomonas vaginalis and amebiasis in warm-blooded animals such as mice and rats when administered at approximately 6 to 18 mg. per kg. of body weight in a single oral dose or in feed. In addition, some of the active components provide control of Eacherichin coli infections in chicks when administered at about 40 mg. per kg. of body weight in a single oral dose. The present compositions show good anticoccidial activity when administered at from about 5.0 p.p.m. to 250 p.p.m. in the diet and are particularly effective against Eimeria tenella, Eimeria brunettz', Eimeria maxima and Eimeria necatrix as shown hereinafter. On a mg./ kg. basis this range is equivalent to about 0.6 to 30 mg./kg.

DETAILED DESCRIPTION The following examples are provided for illustrative purposes and may include particular features of the invention; however, the examples should not be construed as limiting the invention, many variations of which are possible without departing from the spirit or scope thereof. Parts are by weight unless otherwise indicated.

EXAMPLE 1 Preparation of 2-(2-Amino-5-thiadiazolyl) 1methyl-S-nitroimidazole A mixture of 4.71 gm., 0.03 mole, of l-methyl-2-hydroxymethyl-S-nitroimidazole, and 13.3 gm., 0.03 mole, of lead tetraacetate in 200 ml. of benzene, is refluxed while stirring magnetically for about 18 hours, cooled and filtered. The filtrate is washed with 50 m1. of saturated sodium carbonate solution. The organic phase is then separated, and the aqueous phase extracted twice with 30 ml. of chloroform. The combined organic phase is then dried over magnesium sulfate. After filtering and evaporating the organic phase to dryness, the filtrate gives 4.2 gm. of pale yellow 1methyl-5-nitro-2-imidazolecarboxaldehyde, which is dissolved in 25 ml. of hot ethanol, then added to 2.5 gm. of thioscmicarbazide in 20 ml. of boiling ethanol containing two drops of concentrated hydrochloric acid. The mixture is then boiled for a few minutes with stirring, cooled and bright yellow crystals of the thiosemicarbazone of the above aldehyde are collected. The process yields 5.3 gm., which constitutes a 774.0 overall yield, said material having a melting point of 227 C. (decomposition).

To 25 ml. of hot water containing 5.7 gm., of ferric ammonium sulfate dodecahydrato, 2.68 gm. of the above thiosemicarbazone is added, and the mixture is stirred magnetically in a boiling water bath. After 1 hour, an additional 75 ml. of hot water containing 17.1 gm. of ferric ammonium sulfate dodecahydrate is added to the above mixture. The mixture is then heated for approximately 3 hours in a boiling water bath, and filtered while still hot, yielding orange brown crystals which are washedl thoroughly with hot water. The yield is 2.7 gm., having a melting point of from 259 to 260 C. (decomposition). This product is dissolved in about 20 ml. of hot dimethylformamide, filtered and the warm filtrate poured on ice.. The precipitated product is washed thoroughly first with water, and then cold acetone, giving a yellow product: which is dried in vacuo at 100 C. for several hours. The purified product weighs 1.55 gm., and has a melting point of from 268 to 270 C. (decomposition). The product is then submitted to analysis; calculating firm carbon, hydrogen, nitrogen and sulfur, the actual values agreed! closely with the theoretical values.

EXAMPLE 2 A suspension of gm. of ferric ammonium sulfate dodecahydrate in 100 ml. of water is warmed on a steam bath until the solid is completely dissolved. To the above solution 9.6 gm. 0.039 mole, of I-methyl 5 nitro-Z- imidazolecarboxaldehyde 4' methyl 3 thiosemicarbazone is added, and stirred on the steam bath for an additional 90 minutes. A warm solution containing 59 gm., 0.122 mole of ferric ammonium sulfate dodecahydrate in 300 ml. of water is added to the above mixture. The mixture is stirred on the steam bath for an additional 2 hours and filtered. The precipitate is washed with water and acetone to give 6.9 gm. of a yellow solid having a melting point of from 230 to 238 C. Recrystallization from 700 ml. of hot ethyl acetate gives 3.3 gm. of bright yellow needles having a melting point of 218 C.

EXAMPLE 3 Preparation of 2-(Z-Dimethylamino-5-thiadiazolyl)- l-methyl-S-nitroimidazole A mixture of 14.1 gm., 0.1 mole, of 1,2-dimethyl-5- nitroimidazole, 12 -gm., 0.1 mole, of selenium dioxide and 100 ml. of diethyleneglycol dimethyl ether is refluxed while stirring magnetically for approximately 4 hours. The mixture is then cooled and filtered through a layer of diatomaceous earth into a warm solution of 11.5 gm., 0.1 mole of 4',4-dimethyl 3' thiosemicarbazide in 50 ml. of water and 10 ml. of glacial acetic acid. A precipitate forms immediately. The mixture is cooled and filtered to give 7.5 gm. of yellow crystals, which have a melting point of from 205 to 210 C. The crystals are recrystallized twice from methyl cellosolve and once from ethanol, to give 1.3 gm. of orange needles having a melting point of from 208 to 210 C. (decomposition).

A warm suspension of 9.2 gm. of 1-methyl-5-nitro-2- imidazolecarboxaldehyde 4',4 dimethyl 3' thiosemicarbazone (prepared above) in 100 ml. of water is added to a warm solution of 69.4 gm. of ferric ammonium sulfate dodecahydrate in 400 ml. of water. The mixture is magnetically stirred on a steam bath for approximately 4 hours, and filtered. The precipitate is washed with water and acetone giving 8.6 gm. of a brown solid having a melting point of from 238 to 240 C. (decomposition). Two recrystallizations from N,N-dimethylformamide gives 3.6 gm. of yellow crystals having a melting point of 252 C.

EXAMPLE 4 Preparation of 2-(2 Ethylamine 5 thiadiazolyl)-1- methyl 5 nitroimidazole and 2-(2-Dimethylamino-5- thiadiazolyl) -1-ethyl-5-nitroimidazole The ethylamino compound is prepared by employing the procedure set forth in Example 1, with the exception that ethylthisemicarbazide is substituted for thiosemicarbazide. The compound melts at 214-216 C.

With regard to the dimethylamino compound, said compound is prepared by employing the procedure set forth in Example 3, with the exception that l-ethyl 2 methyl- S-m'troimidazole is utilized instead of 1,2-dimethyl-5-nitroimidazole. The process yields l-ethyl 5 nitro2imidazolecarboxaldehyde, 4,4 dimethyl 3' thiosemicarbazone, which is cyclized as in Example 3 to 2-(2-dimethylamino-S-thiadiazolyl -1-ethyl-5-nitroimidazole.

EXAMPLE 5 Preparation of 2-(2-Formamido-S-thiadiazolyl)- l-methyl-S-nitroimidazole The above compound is prepared by refluxing 8 gm. of 2-(2-amino 5 thiadiazolyl) 1 methyl 5 nitroimidazole in ml. of 98% formic acid for 10 hours,

cooling, and adding the mixture to saturated sodium bicarbonate solution, and the solid collected. The compound melts at 225 C. to 227 C.

EXAMPLE 6 Preparation of 2-(2-Acetamido-5-thiadiazolyl)- 1-methyl-5-nitroimidazole The above compound is prepared by heating under reflux for 30 minutes a mixture of 14 gm. of 2-(2-amino- 5-thiadiazolyl) 1 methyl 5 nitroimidazole in 280 ml. of acetic anhydride. The mixture is evaporated to dryness, and the solid residue is Washed thoroughly with ether giving 16.4 gm. of a yellow solid having a melting point of 235 C. (decomposition).

EXAMPLE 7 Preparation of 1-(2-Acetoxyethyl)-5-nitro-2-imidazolecarboxaldehyde A 6.27 gm. portion of 1-(Z-acetoxyethyl)-2-hydroxymethyl 5 nitroimidazole is refluxed with 13.3 gm. of lead tetraacetate in 200 ml. of benzene for 18 hours, cooled, and filtered. The filtrate is washed with 50 ml. of saturated sodium carbonate solution, and the organic phase is separated therefrom. The remaining aqueous phase is twice extracted with 30 ml. of chloroform, and then combined with the above separated organic phase. The combined organic phases are dried under magnesium sulfate, and filtered. The organic solvents are then re moved in vacuo, giving the above aldehyde.

EXAMPLE 8 Preparation of 2-(2-Amino-5-thiadiazolyl)-1-(2- hydroxyethyl)-5-nitroimidazole A 14.25 g. sample of the aldehyde prepared in Example 7 is treated with 5.72 g. of thiosemicarbazide in 150 ml. of ethanol containing a drop of concentrated hydrochloric acid and the mixture is heated on a steam bath for 20 minutes. The hot solution is filtered to remove insoluble materials, cooled and the yellow-brown crystals are collected. The yield of 1-(2-acetoxyethyl) 5 nitro- 2-imidazolecarboxaldehyde thiosemicarbazone is 18.8 g. after drying in a vacuum oven at 60 for 2% hours. Recrystallization of the product gives a yellow solid, melting point 181-1835 C.

The thiosemicarbazone (12 g.) is added to 77 g. of ferric ammonium sulfate dodecahydrate in 500 ml. of water at 60 and the mixture is heated to 90100 for 4 hours. The mixture is cooled, the solid collected and washed with water. Only 0.92 g. (melting point 249- 251) of product is soluble in a large volume of acetone. The remaining product is dissolved in ml. of dimethylformamide, filtered and the filtrate evaporated to dryness to give a solid. This solid is treated with about 20 ml. of acetone, slurried, cooled and collected to give yellow crystals. After drying in an air stream overnight, 5.5 g. (melting point 253.5-255) of 2-(2-amino-5-thiadiazolyl) 1 (2 hydroxyethyl) 5 nitroimidazole is obtained; no carbonyl absorption band is present in the infrared spectrum. Thus, it is not necessary to perform a separate hydrolysis step for the removal of the acetyl group.

EXAMPLE 9 Preparation of 2-(2-Dichloroacetamido-5thiadiazolyl) l-methyl-S-nitroimidazole Five grams of 2-amino 5 (l-methyl 5 nitro-2- imidazolyl) 1,3,4 thiadiazole is added to 25 ml. of dichloroacetic anhydride, and the mixture boiled gently for a few minutes. After standing at room temperature for four hours, 200 ml. of diethyl ether is added, and the mixture stored at 10 C. overnight. The solid is collected, dried, and recrystallized from a mixture of 300 ml. of ethanol and 50 ml. of 2-methoxyethanol to yield 4.5 g. of pure product melting at 246-247 C.

EXAMPLE Preparation of 2-(2-Amino-5-oxadiazolyl)-l-methyl-5- nitroimidazole Method A.In 180 ml. of hot water, 9.3 grams (0.06 mole) of 1- methyl-S-nitro-2-imidazolecarboxaldehyde is slurried while 6.7 grams (0.06 mole) of semicarbazide hydrochloride is added portion wise. After minutes of heating, the mixture is cooled in a refrigerator overnight. The solid is then collected and washed with water and methanol, respectively, to give a yellow product, melting point 272-273 C. (dec.). After drying at 100 for 2 hours under reduced pressure, 11.94 grams of l-methyl- 5-nitro-2-imidazolecarboxaldehyde semicarbazone is obtained. This semicarbazone (6.35 grams or 0.03 mole) is added to 10 grams of anhydrous sodium acetate in 50 ml. of glacial acetic acid and 1.25 ml. of bromine is added with continuous stirring. The mixture is heated gradually to give a nearly clear red solution at 50 C. which becomes increasingly turbid with time. After heating at 75 i3 for 3 hours, the mixture is cooled and poured on ice. The yellow solid is collected, washed with water, then with methanol, and dried under reduced pressure at 70 C. for 3 hours to give 5 grams of crude 2-amino5-(1- methyl-5-nitro-2- imidazolyl) oxadiazole, melting point 284287 C. (dec.). This material is dissolved in boiling dimethylformamide, ethanol added, and the mixture cooled to give yellow crystals, melting point 291293 C. (dec.).

Method B.In 25 ml. of methanol, 0.93 grams (5 mole) of 1-methyl-5-nitro-2-imidazolecarboxylic acid hydrazide and 0.53 grams (5 mmole) of cyanogen bromide are refluxed for 2 hours, cooled, and poured on ice to give a pale yellow solid. This solid is collected, washed with water, and dried under reduced pressure at 100 C. for 2 hours to give 0.65 grams, melting point 286288 C. (dec.), of 2-amino-5-(l-methyl-5-nitro-2-imidazolyl)oxadiazole.

EXAMPLE 11 Preparation of 2-(2-Methylamino-5-oxadiazolyl)-1- methyl-5-nitroimidazole To a solution of 8.5 grams (0.055 mole) of l-methyl- 5-nitro-2-imidazolecarboxaldehyde in 50 ml. of ethanol is added a solution of 5.0 grams (0.056 mole) of 4-methylsemicarbazide in 25 ml. of ethanol and 10 ml. of water containing 2-4 drops of concentrated hydrochloric acid. The resulting solution is heated at 60 -70 C. until a yellow solid starts to separate and then stored at 0 C. for 1 hour; 12.2 grams (98%) of 1-methyl-5-nitro-2-imidazolecarboxaldehyde 4 methylsemicarbazone is obtained melting at 221-223 C.

Seven grams (0.031 mole) of l-methyl-S-nitro-Z-imidazolecarboxaldehyde-4-methylsemicarbazone is dissolved in 85 ml. of glacial acetic acid, 16 grams of anhydrous sodium acetate added, and a solution of 2.5 ml. of bromine in 20 ml. of glacial acetic acid added. The resulting mixture becomes a clear solution as it is heated at 70-80 C. for 2-4 hours. The solvent is evaporated under reduced pressure and the residue treated with shaved ice until a yellow suspension is obtained. The product is collected, washed with cold water, methanol, and finally with ether. Recrystallization from ethanol containing N,N-dimethylformamide affords 2.7 grams (39%) of a yellow product melting at 23924l C. In another preparation, a 63% yield of product melting at 237239 C. is obtained without recrystallization.

EXAMPLE 12 Preparation of 2-(Z-Dimethylamino-S-oxadiazolyl)-1- methyl-S-nitroimidazole A solution of 780 mg. (5 millimoles) of l-methyl-S- 8. nitro-2-imidazolecarboxaldehyde in 10 ml. of of ethanol is treated with 520 mg. (5 millimoles) of 4,4-dimethylsemicarbazide and one drop of concentrated hydrochloric acid to give a yellow solid. The mixture is heated at 40 C. for 5 minutes, cooled to 0 C., and 1180 mg. (98%) of crystalline 1-methyl-S-nitro-Z-imidazolecarboxaldehyde 4,4-dimethylsemicarbazone is obtained. Recrystallization from ethanol containing N,N-dimethylformamide affords 900 mg. (74%) of material melting at 206208 C.

The procedure of Method A of Example 10 is followed except that 7.5 grams (0.31 mole) of l-methyl-5- nitro-2-imidazolecarboxaldehyde 4,4 dimethylsemicarbazone, prepared above, is used. The product is recrystallized from ethanol to give 5.2 grams (69%) of material melting at 180l82 C.

EXAMPLE 13 Preparation of 2-(2-Acetamido-5-oxadiazolyl)-1-methyl- S-nitroimidazole Four grams of acetyl chloride are slowly added to 3.6 grams (0.017 mole) of 2-(2 amino 5 oxadiazolyl) lm-ethyl-S-nitroimidazole suspended in a mixture of 44 ml. of pyridine and 35 ml. of benzene. The addition is completed in 30 minutes, the mixture heated at 6070 C. for 10 minutes, and then poured into 500 ml. of ice and water. After the resulting mixture is stirred for 1 /2 hours, the product separates. Recrystallization from 150 ml. of acetone containing some N,N-dimethylformamide affords 2.4 grams (56%) of pale yellow product. A second recrystallization from acetone gives 2.1 g. of product, melting point 224225 C.

EXAMPLE 14 Preparation of 2-{2-[(Dimethylaminomethylene)amino] -5-thiadiazolyl}-1-methyl-5-nitroimidazole Phosgene gas is bubbled into 100 ml. of N,N-dimethyl formamide at 5-10 C. until 2.0 g. (0.02 mole) is absorbed and a crystalline suspension is formed. This suspension is added in portions to a stirred mixture of 4.5 g. (0.02 mole) of 2-(2-amino-5-thiadiazolyl)-l-methyl-5-nitroimidazole and 100 ml. of N,N-dimethylformamide at 25 C. After 30 minutes the reaction mixture is diluted with 200 ml. of diethyl ether, and the pale yellow solid is collected, washed with ether, and dried. Treatment of this material with ml. of water and drying affords 5.0 g. (89%) of brilliant yellow solid melting at 230-232 C.

EXAMPLE 15 Preparation of 2-[2-(4-Carbethoxy-l-piperazinyl)-5- thiadiazolyl] 1 -methyl-5 -nitroimida.-zole The compound 1-methyl-5-nitro-2-imidazole carboxaldehyde-4-carbethoxy-l-piperazine thiocarbohydrazone is prepared by the procedure of Example 3, 23.2 gm., 0.1 mole, of 4-carbethoxypiperazine 1 thiocarbohydrazide replacing the 4,4-dimethyl-3-thiosemicarbazide. The yield is 8.6 gm. and the melting point 182l83 C.

A suspension of 6.4 gm., 0.0174 mole, of l-methyl-S- nitro-2-imidazolecarboxaldehyde 4 carbethoxy-l-piperazine thiocarbohydrazone (prepared above) in 200 ml. of boiling ethanol is stirred as a solution of 35 mg. of ferric ammonium sulfate dodecahydrate in 200 ml. of hot water is added in one portion. A deep red-brown solution results. After stirring and heating on the steam bath for four hours, a precipitate is present. This is collected, washed with water, dried and recrystallized from hot ethanol to give 3.6 grams of the title compound melting at 179-181 C.

EXAMPLE 16 Preparation of 2-[2- (Hydroxyethylamino)-5- thiadiazolyl] -1-methyl-5-nitroimidazole A solution of 1.3 gm., 0.0058 mole, of 2-[-amino-5- thiadiazolyl)-l-methyl-Smitroimidazole is dissolved in 25 ml. of concentrated hydrochloric acid, cooled to C., stirred, and treated during five minutes with a solution of 0.5 gm., 0.0073 mole, of sodium nitrite in 2 ml. of water. The mixture is kept at room temperature for eighteen hours. The precipitate present is collected, washed with water, dried and then extracted with warm acetone. Removal of the acetone leaves a solid residue which is recrystallized from a mixture of acetone and diethylether to give 0.16 gm. of 2-(2-chl0r0-5-thiadiazolyl)-1-methyl-5-nitr0imidaz0le as yellow crystals, melting at 135137 C.

A mixture consisting of 4.9 gm., 0.02 mole, of 2-(2- chloro-5-thiadiazolyl)-1-methyl 5 nitroimidazole, 3.0 gm., 0.05 mole, of ethanolamine, and 50 ml. of p-dioxane is stirred at room temperature for twenty-four hours. The precipitate is collected, washed with aqueous sodium bicarbonate solution, dried and recrystallized from methanol to give the pure compound melting at 208-209" C. Working up the mother liquors gives additional material, the total yield being 3.4 gm.

EXAMPLE 17 Preparation of 2-[2-(3-Dimethylaminopropylamino)-5- thiadiazolyl] -1-methyl-5-nitroimidazole A mixture consisting of 3.8 gm., 0.0155 mole of 2-(2- chloro-5-thiadiazolyl)-1-methyl 5 nitroimidazole, prepared as in Example 16, 2.5 gm., 0.029 mole of 3-dimethylaminopropylamine, 2.1 gm. 0.025 mole, of sodium bicarbonate and 100 ml. of benzene is refluxed for twentytwo hours. The reaction mixture is cooled to room temperature, washed with aqueous sodium bicarbonate and sodium chloride solutions. Cooling to 5 C. gives a crystalline precipitate which is collected and recrystallized from benzene to give the pure compound melting at 153-154 C. More product is obtained from the mother liquors to give an overall yield of 3.0 gm.

EXAMPLE 18 Preparation of 2-(2-Amino-5-oxadiazolyl)-1-(2-hydroxyethyl) 5 nitroimidazole, 2-(2-methylamino-5-oxadiazolyl)-1-(2-hydroxyethyl)-5-nitroimidazole, and 2-(2- dimethylamino 5 oxadiazolyl)-1-(2-hydroxyethyl)- S-nitroimidazole 1-(2-hydroxy)-5-nitro 2 imidazolecarboxaldehyde (1 mole) is treated with 1 mole of semicarbazide hydrochloride in ethanol to give nearly a quantitative yield of semicarbazone. The product is treated with bromine and sodium acetate in the manner described in Method A of Example 1 to give the first compound above which melts with decomposition at 228230 C.

The use of 4-methylsemicarbazide and'4,4-dimethylsemicarbazide hydrochlorides instead of semicarbazide hydrochloride, followed by treatment of the methylated semicarbazones with bromine and sodium acetate as above, gives, respectively, 2-(2-methylamino 5 oxadiazolyl)-1-(2-hydroxyethyl)-5-nitroimidazole, melting at 202.5 204 C., and Z-(Z-dimethylamino-S-oxadiazolyl)- 1-(2-hydroxyethyl)-5-nitroimidazole, melting at 171- 173 C.

EXAMPLE 19 Preparation of 2 (2-Amino-5-thiadiazolyl)-1- (Z-acetoxyethyl) -5-nitroimidazole The sample (0.1 g.) of 2-amino-5-[1-(2-hydroxyethyl)-5-nitro-2-imidazolyl]thiadiazole is dissolved in 2 ml. of hot glacial acetic acid and a drop of concentrated sulfuric acid is added. The solution is refluxed for 45 minutes, cooled and poured on ice to give a yellow solid. This solid is collected, washed with water and dried; the yield is 0.1 g., melting point 159-162 C. (turbid). A purified sample of the 2-amino-5-[1-(2-acetoxyethyl)-5- nitro-2-imidazolyl]thiadiazole, melts at 164165.5 C.

10 EXAMPLE 20 Preparation of 2-(2-Acetamido-5-thiadiazolyl)-1- (Z-acetoxyethyl)-5-nitroimidazole The sample (0.1 g.) of 2-amino-5-[1-(2-hydroxyethyl)-5-nitro-2-imidazolyl]thiadiazole is added to 1.5 ml. of acetic anhydride and heated under reflux for 20 minutes. After cooling, the mixture is evaporated to dryness to give a tan solid which is slurried with ether and collected, melting point 258265 C.; the yield is 0.11 g. This solid is recrystallized from acetone to give the purified product, melting point 264-268 C.

EXAMPLE 21 Preparation of 2-(2-Amino-5-thiadiazolyl)-1- methyl-S-nitroimidazole Hydrochloride Four grams of 2-(Z-amino-S-thiadiazolyl)-l-methyl-5- nitroimidazole is added to concentrated hydrochloric acid. The resultant precipitate of the hydrochloride is filtered off and air dried; melting point 249 C. with decomposition. When the hydrochloride is added to water, the free base is formed again without the necessity of using alkali.

EXAMPLE 22 Preparation of 2-(2-Hydroxymethylamino-5-thiadiazolyl -1-methyl-5-nitroimidazole A suspension of 5 g. of 2-(2-amino-5-thiadiazolyl)-1- methyl-S-nitroimidazole in 200 ml. of 36% aqueous formaldehyde solution is stirred for 20 hours at room temperature. The suspension is cooled in ice, and the insoluble solid is collected by filtration and washed with acetone. The yield of the title compound is 6.15 g., melting point 177 C. with decomposition (rapid heating).

EXAMPLE 23 Preparation of 2-(Z-Amino-S-thiadiazolyl)-1-ethyl-5- nitroimidazole A slurry of ;9-(1-ethyl-5-nitro-2-imidazolyl)styrene (42.7 g. or 0.175 mole) in 350 ml. of methanol containing 14.6 ml. of water at 25 C. is treated with ozone until a nearly clear, pale-yellow solution is obtained. Subsequently, the mixture is treated with 42 g. of sodium iodide in 138 ml. of water and 20.3 ml. of glacial acetic acid at 25 C. The mixture is stirred for 40 minutes and 44.1 g. (0.288 mole) of sodium thiosulfate in 242 ml. of water added. The mixture is filtered, and the filtrate concentrated at 7075 C. under 15-20 mm. of pressure to give 450 ml. of solution. The solution is acidified with 50 ml. of 6N hydrochloric acid and the benzaldehyde removed at 7075 C. under 15-20 mm. of pressure. The residue is then neutralized With saturated sodium bicarbonate solution and extracted with ethyl acetate to give an 81.5% yield of solid aldehyde, melting point 6167 C. after stripping. A sublirned sample of the aldehyde melts at 68-69 C. with softening at 65 C. When the aqueous ozonized solution, after benzaldehyde is removed, is treated with thiosemicarbazide and mineral acid or semicarbazide hydrochloride and heated, the 1-ethyl-5-nitro- 2-imidazolecarboxaldehyde thiosemicarbazone, melting point 241 C., or semicarbazone, melting point 223- 226 C., is obtained. The compound p-(1-ethyl-5-nitro-2- imidazolyl)styrene is prepared by treating 1-ethyl-2- methyI-S-nitroimidazole with benzaldehyde in absolute ethanol and potassium tertiary butoxide in nitrogen atmosphere below 37 C. A sample purified from 95% ethanol melts at 136.5137.5 C.

A solution of 184 g. of ferric ammonium sulfate dodecahydrate in 680 ml. of water is heated to 60 C. and 23.1 g. of 1-ethyl-5-nitro-2-imidazolecarboxaldehyde thiosemicarbazone (prepared as above) is added with efiicient stirring. The temperature is raised to C. and vigorous stirring continued for 4 hours. The reaction mixture is cooled to 4 C. and filtered. The solid is washed with warm water and dried under vacuum at 110 C. and then extracted with 800 m1. of hot acetone, followed by three 300-ml. portions of hot acetone. Cooling the acetone gives a solid product, which is filtered off; melting point 231233 C. A second crop with the same melting point is obtained by evaporating the filtrate and recrystallizing the residue from acetone. The combined yield is 17.0 g.

EXAMPLE 24 Preparation of 2-(2 Amino--thiadiazolyl)-l-benzyl-5- nitroimidazole 0.5 g. (1.64 millimole) of fi-(l-benzyl-S-nitro-Z- imidazolyl)styrene in 95% aqueous methanol is treated with ozone until a clear solution is obtained. To this solution at C., 0.312 g. (1.64 millimole) of sodium metabisulfite (Na S O in 3 ml. of water is added. The mixture is then evaporated to dryness under reduced pres- :sure at 75 C. and the solid extracted with ethyl acetate. The ethyl acetate extracts are dried, evaporated to drymess to give a yellow-orange residue (probably bisulfite addition product) which solidifies upon standing. This material is dissolved in ml. of aqueous methanol and 0.15 g. of thiosemicarbazide and a drop of 6N hydrochloric are added. After refluxing for 15 minutes and cooling, 0.25 g., melting point 193-196" C., of l-benzyl- S-nitro-2-imidazolecarboxaldehyde thiosemicarbazone is isolated. The use of a half of an equimolar amount of sodium meta-bisulfite affords the title aldehyde instead of the bisulfite addition product. When the ethyl acetate extract is evaporated to dryness and dissolved in 75% aqueous ethanol and semicarbazone is added, and the mixture is heated on a steam bath for 10 minutes, and sodium acetate is added, the semicarbazone derivative, melting point 226228 C. is obtained. Twelve grams of 1-benzyl-5-nitro-2-imidazolecar'boxaldehyde thiosemicarbazone are added to a stirred solution of 76.2 g. of ferric ammonium sulfate dodecahydrate in 600 ml. of water at 50 C. and the slurry is heated at 90-95 C. for 6 hours. The mixture is cooled in ice and the yellowbrown solid collected, washed with water, and dried under reduced pressure. The yield of product is 10.9 g. Recrystallization from methanol gives analytically pure material melting at 2132l5 C.

EXAMPLE 2-(2-Amino-5-oxadiazolyl) -1-ethyl-5- nitroimidazole A slurry of 10.2 g. of 1-ethyl-5-nitro-2-imidazolecarboxaldehyde semicarbazone (prepared as in Example 23), and 15.0 g. of anhydrous sodium acetate in 75 ml. of glacial acetic acid is treated with 1.87 ml. of bromine at 81 C. under agitation. Stirring is continued at 75-80" C. for 17 /2 hours, at which time workup of a sample shows starting material still present. An additional 7.5 g. of sodium acetate and 1.00 ml. of bromine are added and stirring continued for 3 hours. A final 3.85 g. of sodium acetate and 0.50 ml. of bromine are added and the reaction mixture is stirred for one hour at 75 C. The reaction mixture is cooled to 15 C. and poured over ice. The product is filtered off, washed with Water and dried under reduced pressure. The yield is 6.3 g. and the melting point 265 C. with decomposition.

EXAMPLE 26 Preparation of 2-(2-Amino-5-oxadiazolyl)-l-benzyl-5- nitroimidazole Preparation of A slurry of 6.1 g. of l-benzyl-5-nitro-2-imidazolecarboxaldehyde semicarbazone (prepared as in Example 24) and 8 g. of anhydrous sodium acetate in 40 ml. of glacial acetic acid is stirred at 40 C. While 3.4 g. of bromine in 15 ml. of acetic acid is slowly added. The mixture is heated at 6570 C. for 4 hours, then poured on ice and 12 the yellow solid collected, washed with water, and dried under reduced pressure. The yields is 5.7 g. Recrystallization from acetone gives analytically pure material melting at 261.5262.5 C.

EXAMPLE 27 Preparation of 2-[2-(2,2 Diethoxyethylamino)-5-thiadiazolyl] -1-rnethyl-5-nitroimidazole A mixture composed of 9.8 g. of 2-(2-chloro-5-thiadiazolyl)-1-methyl-5-nitroimidazole, (Example 16), 11.0 g. of 2,2-diethoxyethylamine, and 250 ml.of dioxane is stirred and heated on the steam bath for 12 hours. It is then diluted With 500 ml. of cold water, cooled, and the precipitated yellow solid collected and dried. Recrystallization from 50% aqueous ethanol gives the pure compound melting at 154-155 C.

EXAMPLE 28 Preparation of 2-(2-Piperidino-5-thiadiazolyl)- 1-rnethyl-5-nitroimidazole EXAMPLE 29 Preparation of 2- 2-n-Hexylamino-S-thiadiazolyl l-methyl-S-nitroimidazole The preparation of the title compound is carried out essentially as described for the 2-piperidino derivative (Example 28), 6.5 g. of n-hexylamine replacing the piperidine. The crude product is recrystallized from ml. of Z-methoxyethanol to give 5.2 g. of the pure compound melting at 146147 C.

EXAMPLE 3 0 Preparation of 2- [4- (2-Hydroxyethyl) -1-piperaziny1- 5 -thiadiaz0lyl] 1-methyl-5 -nitroimidazole The preparation of the subject compound is carried out essentially as described for the 2-piperidino derivative (Example 28), 3.9 g. of 1-(2 hydroxyethyl)piperazine replacing the piperidine. After recrystallization from ethanol, 3.5 g. of pure compound is obtained, melting at 240- 206 C.

EXAMPLE 31 Preparation of 2-{2- [4- (3-Dimethylaminopropyl) -1-piper1d1no]-5-thiadiazo1yl}-1-methyl-5-nitroimidazole The preparation of the title compound is carried out by the procedure described for the 2-piperidino derivative (Example 28), 5.1 g. of 4-(3-dimethylaminopropyl)-piperidine replacing the piperidine. Recrystallization from rlnlesthaiol gives 5.6 g. of pure product, melting at 174- EXAMPLE 32 Preparation of 2-{2- N- 2-Hydroxyethyl methylamino] 5-th1adiazolyl}-1-methyl-5-nitroimidazole The preparation of the above compound is carried out essentially as described for the 2-piperidino derivative (Example 28), an equivalent of N-met-hylethanolamine replacing the piperidine. The pure compound melts at 158160 C. after recrystallization from methanol.

EXAMPLE 33 Preparation of 2-[2-(1-Piperazinyl)-5-thiadiazolyl]- 1-methyl-5-nitroimidazole The preparation of the subject compound is carried out essentlally as described for the 2-piperidino derivative (Ex- 13 ample 28), two equivalents of piperazine replacing the piperidine. The crude product is recrystallized from 2-methoxyethanol to yield the pure compound melting at 240- 241 C.

EXAMPLE 34 Preparation of 2-[2-(4-Methyl-l-piperazinyl)- S-thiadiazolyl]-l-methyl-S-nitroimidazole The preparation of the above compound is carried out in the manner described for the 2-piperidino derivative (Example 28), an equivalent of l-methylpiperazine replacing the piperidine. The crude product is purified by recrystallization from 2-methoxyet-hanol and melts at 242244 C.

EXAMPLE 35 Preparation of 2-{2- [4-(3-Dimethylaminopropyl)-l-piperazinyl] --thiadiazolyl}-1-methyl-5-nitroimidazole A solution of 2-(2-chloro-5-thiadiazolyl)-1-methyl-5- nitroimidazole in 200 ml. of dioxane is treated with 5.1

g. of l-(3-dimethylaminopropyl)piperazine, and the mixture stirred at room temperature for 18 hours. The precipitate is collected, dissolved in 100 ml. of water, made alkaline with sodium hydroxide solution, and then extracted with 500 ml. of chloroform. The chloroform extract is dried and the solvent removed under reduced pressure. The yellow residue is recrystallized from 100 ml. of ethanol to give 2.9 g. of product, melting at 174- 175 C.

EXAMPLE 36 Preparation of 2-{2- [4- (Z-Pyridyl) l-piperazinyl] 5-t-hiadiazolyl}-l-methyl-S-nitroimidazole The preparation of the above compound is carried out essentially as described for the 2-[4-(3-dimethylaminopropyl)-l-piperazinyl] derivative (Example 35), an equivalent of l-(2-pyridyl)piperazine replacing the l-(3- dimethylaminopropyl)piperazine. After recrystallization from Z-methoxyethanol, the pure compound melts at 280- 282 C.

EXAMPLE 37 Preparation of 2-{2- [4- 8-Thiazolyl) -1-piperazinyl] 5-thiadiazolyl}-l-methyl-S-nitroimidazole The preparation of the subject compound is carried out essentially as described for the 2-[4-(3-dimethylaminopropyl)-1-piperazinyl] derivative (Example 35), an equivalent of 1-(2 thiazolyl)piperazine replacing the l (3- dimethylaminopropyl)piperazine. After recrystallization from 2-met-hoxyethanol, the pure compound melts at 297-300" C.

EXAMPLE 38 Preparation of 2-(2-Cyclohexylamino-5-thiadiazolyl) l-methyl-S-nitroimidazole A mixture of 5.0 g. of 2-(2-chloro-5-thiadiazolyl)-lmethyl-S-nitroimidazole, 6.0 g. of cyclohexylamine and 125 ml. of dioxane is stirred at reflux until thin-layer chromatographic analysis indicates that the chloro intermediate is completely utilized. The dioxane is removed under reduced pressure and the residue triturated with aqueous sodium bicarbonate solution. Recrystallization from a mixture of ethylacetate and acetone yields the pure compound melting at 2l52l7 C.

EXAMPLE 39 Preparation of 2-(Z-t-Butylamino-S-thiadiazolyl l-methyl-S-nitroimidazole The preparation of the above compound is accomplished essentially by the procedure described for the 2- cyclohexylamino derivative (Example 38), an equivalent of t-butylamine replacing the cyclohexylamine. Recrystallization of the crude product from a mixture of ethyl acetate and acetone gives the pure compound melting at 249- l C.

14 EXAMPLE 40 Preparation of 2-(2-n-Octylamino-S-thiadiazolyl)- l-methyl-5-nitroimidazole The preparation of the above compound is carried out essentially as described for the 2-cyclohexylamino derivative (Example 38), an equivalent of n-octylamine replacing the cyclohexylamine. The crude product is purified by recrystallization from aqueous 2-methoxyethanol and then melts at l30132 C.

EXAMPLE 41 Preparation of 2-(2-Chloroacetamido-5-thiadiazolyl)- l-methyl-5-nitroimidazole Twenty grams of 2-(2-amino-5-thiadiazolyl)-1-methyl- S-nitroimidazole is added in portions to a solution of 60 g. of chloroacetic anhydride in 250 ml. of dioxane at 95 C. The mixture is stirred at this temperature for three hours. After standing at room temperature, the mixture is filtered and precipitate washed with diethyl ether and dried. Recrystallization from 2-methoxyethanol gives the pure compound, melting at 239-240 C. with decomposition.

EXAMPLE 42 Preparation of 2- (2-Morpholinoacetamido-5- thiadiazolyl) 1-methyl-5-nitro imid azole EXAMPLE 43 Preparation of 2-(2-Amino-5-thiazolyl)-1-propy1- 5 -nitroimidazole The procedure of Example 23, with the substitution of fi-(l-propyl-S-nitro-Z-imidazolyl)styrene for 8 (1 ethyl- 5-nitro-2-imidazolyl(styrene in the first step is utilized to prepare 2 (2 amino-S-thiadiazolyl)-1-propyl-5-nitroimidazole, melting at 234.5236.

EXAMPLE 44 Preparation of 2- (2-Benzylamino-5-thiadiazolyl) 1-methyl-5-nitroimidazole A solution of 7.4 g. of 2-(2-chloro-5-thiadiazolyl)- l-methyl-S-nitroimidazole in ml. of dioxane is stirred and treated with 7 grams of benzylamine. Stirring is continued at room temperature for 18 hours. The heavy precipitate is then collected, washed with 200 ml. of warm water, and dried. Recrystallization from ml. of boiling Z-methoxyethanol gives 3.4 grams of the title compound, melting at 2l9221 C.

EXAMPLE 45 Preparation of 2-[2-(4-Chlorophenylethylamino)-5- thiadiazolyl)-1-methyl-5-nitroimidazole The preparation of the title compound is carried out essentially as described in Example 28 for the 2-piperidino derivative, 7.5 g. of 4-chlorophenyl-ethylamine replacing the piperidine. The crude product is recrystallized from 15 150 ml. of 2-methoxyethanol to give 6.5 g. of the pure compound melting at l80-181 C.

EXAMPLE 46 Preparation of 2-(2-Allylamino-5-thiadiazolyl)-1- methyl-S-nitroimidazole The preparation of the above compound is carried out essentially as described for the 2-piperidino derivative in Example 28, 4.0 g. of allylamine replacing the piperidine. The crude product is recrystallized from 400 ml. of ethanol plus 25 m1. of acetone to give 5.3 g. of the pure compound melting at 211212 C.

EXAMPLE 47 Preparation of 2-[2-(l-pyrrolidinyl)-5-thiadiazolyl]- l-methyl-S-nitroimidazole The preparation of the title compound is carried out essentially as described for the 2-piperidino derivative in Example 28 5.0 g. of pyrrolidine replacing the piperidine. The crude product is recrystallized from 400 m1. of 2- methoxyethanol to give 6.2 g. of the pure compound melting at 25 8260 C.

EXAMPLE 48 Preparation of 2-[2-(4-Hydroxypiperidino)-5- thiadiazolyl]-1-methyl-5-nitIoimidazole The preparation of the above compound is carried out essentially as described for the 2-piperidino derivative in Example 28, 6.5 g. of 4-hydroxypiperidine replacing the piperidine. The crude product is recrystallized from 200 ml. of 2-methoxyethanol to give 7.3 g. of the pure compound melting at 216218 C.

EXAMPLE 49 Preparation of 2-[2-(2-Morpholino) ethylamino-S- thiadiazolyl-1-methy1-5-nitroimidaz0le The preparation of the above compound is carried out essentially as described for the 2-piperidino derivative in Example 28, 9.0 g. of 2-(2-morpholino)ethylamine replacing the piperidine. The crude product is recrystallized from 125 ml. of Z-methoxyethanol to give 6.4 g. of the pure compound melting at 160l62. C.

EXAMPLE 50 Preparation of 2- I-Imidazolyl) -5-thiadiazoly1]-1- methyl-S-nitroimidazole 2.5 g. of 54.7% sodium hydride in mineral oil dis persion is added to a solution of 4.0 g. of imidazole in 300 ml. of dimethylformamide. The mixture is stirred until hydrogen evolution ceases (about '/2 'hour). 12.3 g. of 2-(2-chloro-5-thiadiazolyl)-l-methyl-S nitroimidazole is then added and the mixture is then stirred at room temperature for 6 hours. 600 m1. of water is added, the precipitate is collected, washed with water, and dried. Recrystallization of the crude product from a mixture of 400 ml. of 2-methoxy-ethanol and 100 ml. of dimethylformamide gives 11.2. g. of the pure compound melting at 251-252 C., with decomposition.

EXAMPLE 51 Preparation of 2- (2-Diethylamino-5-thiadiazolyl)- l-methyl-S-nitroimidazole The preparation of the above compound is carried out essentially as described for the 2-piperidino derivative in Example 28, 5 .0 g. of diethylamine replacing the piperidine. The crude product is recrystallized from 250 ml. of ethanol to give 6.0 g. of the pure compound melting at 1 6 EXAMPLE 52 Preparation of 2- [Z-(Z-Dimethylaminoethyl) amino-5- thiadiazolyl]-1-methyl-5-nitroimidazole The preparation of the title compound is carried out essentially as described for the Z-piperidine derivative in Example 28, 6 g. of 2-dimethylaminoethylamine replacing the piperidine. The crude product is recrystallized from 250 ml. of ethanol to give 3.4 g. of the pure compound melting at -172 C.

EXAMPLE 53 Preparation of 2-[2-(1-Ethyl-3-piperidylamino)-5- thiadiazolyl]-1-methyl-5-nitroimidazole The preparation of the title compound is carried out essentially as described for the 2-piperidino derivative in Example 28, 8.5 g. of 3-amino-1-ethylpiperidine replacing the piperidine. The crude product is recrystallized from isopropanol to give the pure compound metling at 151- 152 C.

EXAMPLE 54 Preparation of 2F[2-(4-Carbethoxy-l-piperazinylacetamido)-5-thiadiaz0lyl]-l-methyl-S-nitroimidazole 6.0 g. of 2-(2-chloroacetamido-S-thiadiazolyl)-1-methy1-5-nitroimidazole is placed in the thimble of a continuous extraction apparatus, which is attached to a threeliter round bottom flask with internal stirrer which contains a solution of 4.0 g. of potassium iodide and 7.0 g. of l-carbethoxy piperazine in one liter of acetone. The mixture is stirred at reflux until all the nitroimidazole derivative is extracted from the thimble (about 20 hours). The acetone is removed under reduced pressure and the residue shaken with 200 ml. of water. The water-insoluble product is collected, dried and recrystallized from. 300 ml. of dioxane to give 7.2 g. of pure compound melting at 215217 C.

EXAMPLE 55 Utilization of compounds of the present invention in controlling Trichomonas vaginalis infections This example demonstrates, employing two modes of administration, the efi-lcacy of 2-(2-amino-5-thiadiazolyl)- l-methyl-S-nitroimidazole against T richomonas vaginalis infections.

A. The first mode of administration, hereinafter designated Test A, employs six groups of mice, three groups of five and three groups of ten. The mice are inoculated with 50,000 culture-derived Trichomonas vaginalis (Thoms strain). The above compound is thoroughly mixed into ground feed and presented to the mice one day after inoculation. The average ad libitum drug intake, that is milligrams per kilogram of body weight per day, is estimated from the drug diet intake for 5 full days, and mouse weights taken just before and just after treatment.

B. The second mode of administration, hereinafter designated Test B, employs six groups of mice, five groups of ten and one of five. The mice are inoculated with 50,000 culture-derived Trichomonas vaginalis (Thoms strain). The above compound is administered in a single oral dose by gavage one day after inoculation.

Six days post inoculation scrapings, from the subcutaneous sites of inoculation, are searched microscopically for motile trichomonads, and antitrichomonal activity is concluded to be present in those instances where motile trichomonads are eliminated from lesions present at the site of inoculation.

Mouse body weight gain from day 1 to day 6 postinoculation shows that 2-(2-amino-5-thiadiazolyl)-lmethyl-S-nitroimidazole is well tolerated by the animals ingesting same. Furthermore, experimentation demonstrates that more than 1,000 mg. per kilogram of body weight of said compound is well tolerated.

17 The results of the test are set forth in the following tables:

TABLE I.-TEST A Total Dose, mice Percent Compound mg. tested Cleared cleared 2-(2-amino-5-thiadiazoly1)-1- methyl-5-nitroimidazole 170 5 5 100 1 Dose is in terms of milligrams per kilogram of body weight per day.

TABLE II.-TEST B 1 Dose is in terms of milligrams per kilogram of body weight.

C. Female albino mice (ICR strain) were inoculated subcutaneously with approximately 50,000 Trichomonas vaginalis ('Il1oms strain) harvested from a 24 hour culture. The mice were allocated twenty-four hours postinoculation to ten-mouse test groups, weighed, and subjected to treatment. The present compounds were administered orally; either in the diet for five days or in a single gavage dose, and intakes were estimated from average mouse weights, feed intakes, or both. Sham-dosed controls were included in every assay. Six days postinoculation, microscope examination of tissue debris from the lesion at the site of inoculation in untreated mice revealed motile trichomonads. Neither the parasites nor the lesions could be detected in mice which received adequate treat ment. In the latter case, saline washings of the sites of inoculation were examined for trichomonads. A regimen was considered active if at least 50% of the mice treated harbored no motile trichomonads. The following Table III summarizes the results of the testing.

TABLE III.-TE ST 0 Number cleared lnmnber tested Dose, a/ 5- 100 50 25 12 2-[2-(2-morpholinoacetamido)-5- thiadiazolyll-l-methyl-S- nitroimldazole 2-(2-amino'5-thiadiazo1yl)-1-metl1yl- 5-nitroimidazole hydrochlorlde.. 10/10 2-(2-ethylamlno-5-thiadiazolyl)-1- methyl-5-nitrolmidazole 4/5 2-(2-[4-(3-dimethylaminopro yl)-1- piperazinyl]5thiadiazolyl -1- -methyl-5-nitro1midazole 18/20 2-[2-(1-p1perazinyl)-5-thiadlazolyl]- l-methybfi-nitroimidazole 2-l2-[4-(3dimethy1aminopropyl)-1- plperidinol-s-thiadiazolyll-lmethyl-S-nitrolmidazole 14/14 2-[2-(2,2-diethoxyethylamino)-5- thladiazolyH-l-methyl-S- nitmimirlamle 2-(2-al1ylamino-5-tldadiazolyl)-1- methy1-5-nitroim1dazole 2-[2-(l-pyrrolidinyl)-5-tl1lad1azolyl]- l-methyl-S-nitromidazole 2-[2-(4-hydroxypiperidino)-5- thiadiazolyllmethyl-S- nitroimidazolefl 2-[2-(2-morpholino)-ethylamino-5- thladiazolyH-l-methylnitroimidazole 5/5 9/10 2-(2-diethylamino-5-thiadla lyl) methyl-.Smltroimidazole 2-[2-(2-dimethylamm0ethyl)amino- 5-thiadiazolyll-l-methyl-5- nitroimidmnle 2-[2-(1'ethyl-3- iperldylam1no)-5- thiadiazolyl -1-methyl-5- nitroimidazole 2-[2-(4-carbetl1oxy-1-piperazmylacetamido)-5-thiadiazo yl]-1- methyl-fi-nitroimidazole EXAMPLE 56 Utilization of compounds of the present invention in controlling coccidiosis The anti-coccidial activity of the compounds of the invention is demonstrated by the following tests; one-day old Peterson cross cockerels of approximately equal size and weight, were divided into groups and the groups placed in separate cages with wire floors. A broiler feed diet of the formulation set forth below and having graded levels of test medicament intimately blended therewith was prepared and administered to the various groups. The formulation used was as follows:

The medicated and unmedicated diets were presented to TABLE rv oontinued the chicks and the'chicks were perm tted to feed and b drink ad libirum from two days prior until 7 days followfififfj; g gg Perc'ent mg the oral inoculation with sporulated oocysts of Elm- Structure in diet treated survlval eria tenella. The number of oocysts inoculated directly 5 '2 I24(dtnethylaminomethylem mto theorem of all chicks 1n the test was sufliclent to proangiraql-s-th ad o y l- -m y d e 85100% mortality in the untreated controls. The o 20 40 quantity necessary to produce this mortality rate was de- 1%? 5 12% termined prior to the time of inoculat on by glvrng graded 2 [2 (4 pipeminy1) 5 thiadiam1yn 1 q a tltles of oocysts to comparable blIdS. Seven days fol- 1O ethyl-fi-nitroimidazole g 4g gg lowing moculauon, the test was terminated and the mor- 2 (2 mino 5 thiadiaz01y1)qgthywtahty rate recorded for each group, nitri midazole 15 10 100 The results obtained are provided in Table 1V where 'f$$fii ifffif'g'ff ffki 250 20 1t can be seen that the compounds of this invention are -(2 gnr1ino5-thiadiazo y Y 250 5 20 1'11 I'Olml 8.20 e highly effective as ant1-cocc1d1al agents. 15 2-(2-hydroxymethylamino-S-thiadiflzoyl) -1-meth yl-5-nitroimidazole- 250 5 100 60 70 2-{ 2-[N-(2-hydroxyeth yl) -methy1- amino1-5-thiadiazolyl )-1-methyl-5- TABLE IV nitroimidazole lgg g 128 1: t N 2(2-morpholinoacetamido-S-thiadiali ni gfi P t 20 zoyl):1-methyl-5-nitroimidazole 125 5 100 Structure in diet treated survival 2-(2;an11no-5-thiadiazolyi)-1-methy1-5- mtrounidazole hydrochloride 60 10 100 2-(2-amino-5-thiadiazolyl)1-methyl- 2-(2-benzylamino-5 thiadlazolyl)-1- 5-nitroimidazole 0 39 methyl-5-nitroim1dazole 60 10 90 125 10 100 2-{2-(4-methyl-1-piperaz1nyi:5-)

60 10 70 thiadiazolyll-l-methyl-fi-mtroso 10 imidazole 125 v 5 so 2-(Z'aeetamido-5-thiadiazolyl)-1- 2-'{2-H(3-dimethyla1ninopropyl)-1- methyl-5-nitroimidazoie 0 20 15 p1perazinyll-5-th1ad1azolyl-l1- 125 10 100 methyl-fi-nitroimidazole 125 5 40 2(2 011 1501101 11 1 60 10 100 -me yamno-- a azoy methyl-fi-nitroimidazole 0 40 12 EXAMPLE 57 125 10 100 g8 1g 28 Test 1n mice against T rypanosoma cruzl !Q'g' F- 0 20 10 Tests for chemotherapeutic activity were conducted in m y mum awe 125 10 m0 1 3 female mice infected expe y Wlth 2% g 28 B-strain Trypanosama cruzi. Mice weighing about 1 8 to 2-(2-fonnamldo-5-thiadlazolyl)-1- 22 grams were inoculated subcutaneously wlth approxlmethyl-5-nitwimidawle i8 53 mately 100,000 parasites obtained from the blood of 60 10 B0 donor mice. Treatment in the diet was administered from 30 day 6 through day 12 postinoculation. Infected uninocu- 2- Z-amin -5 (11 11-1- th lrutroiIn da fia-. gi 321... 0 20 15 40 lated controls were included 1n each experunent. The C1!- 500 5 100 2 (dimethylammomadiMOM) 1 terion of chemotherapeutic activrty was prolongation of methyl-fi-nitroimidazole o 20 40 survival of tested treated mice relative to controls. The

250 5 results are summarized in the following Table V:

TABLE V Percent diet Median Percent concen- Approx survival survivors tration, imate time, days 30 days postinocmg./kg./ (treated! (treated/ Compound ulation day controls) controls) 2-[2-(4-carbethoxy-l-piperazinyl)-5-thiadiazolyl]-1-methyl-5-nitroimidazole 0. 01 15 30/16 90/20 2-(Z-dimethylamino-E-thiadiazolyl)-1-metl1yl-5-nitroimidazole 0. 01 13 30/16 70/20 0. 01 12 30/14 70/0 0. 005 5 a0/14 /0 2-2-[(dimethylaminomethylene)amin01-5-tl1iadiazoly1-1-1nethy1-5-nitroin1idazole 0. 01 15 30/17 100/40 0. 01 12 30/14 90/0 0. 005 5 16/14 40/0 0. 0025 2. 7 21/14 40/0 2-(2-amino-5-oxadiazolyl)-1-methyI-5-nitroimidazolo.. 0. 01 16 30/l7 10 /2 0. 005 7 30/17 /0 0. 0025 3 30/15 50/0 2-(2-aeetamido-5-oxadiazoly)-1-metl1y1-5-n1troimidazole..-.. 0. 01 12 30/ 16 7 /2 0. 01 14 a0/14 50/0 I 0. 005 5 19/14 30/0 2-(2-amlno-5-thiadlazolyl)-1-(2-hydroxyethyl)-5-nitroimidazole 0. 01 14 30/16 90/20 2-(2am1no-5-th1adiazolyl) -1-ethy1-5-n1trnirnlda7nla 0, 01 15 3O/I17 69/20 2-[2-(2-morpholinoacetamido)-5-thiadia2olyl]l-methyl-5-nltroimiaz0le 0. 01 12 30/17 50/20 0. 01 11 30/14 50/0 2-(2-amino-5-tl1iadiazolyl)-1-methyl-5-nitroin1idaz0le hydrochloride 0. 01 15 8 /15 0. 01 12 30/18 /0 0.0075 s 22/15 30/0 0.0075 9 25 18 40/0 7 0.005 5 30/15 50/0 2-{2;[4-(3-dimethylaminopropyl)-1-piperazinyl]-5-thiadiazolyl}-1-methyl-5- mtroimidazole 0.01 14 30/l7 70/20 0 0025 3 3D/15 50/0 21 EXAMPLE 58 Tests to determine intestinal amebiasis in rats Female weanling rats (Wistar strain from Royalhart Farms) were inoculated intracecally during laparotomy with 100,000 to 200,000 amebas from a 48-hour culture; a method first reported by Jones, 1946 (Ann. Trop. Med. Parasit. 40(2): 130-140). The rats were divided by random selection into groups with ten animals each; one group remained as the untreated control. Treatment by drug-diet mixture was begun on the day of inoculation and continued for five days. The animals were necropsied and examined for infection on the sixth day. The infection was evaluated by assessing the degree of infection for each animal and the average degree of infection, i.e., A.D.I., for each group, using a scoring system similar to that described by Jones, 1946. The percent of the A.D.I. suppression relative to the untreated control A.D.I. (Abbotts formula) was used to evaluate the activity of candidate compounds. The following Table VI summarizes the results.

TAB LE VI Approxi- Percent Percent mate suppresdrug dosage sion of concen- (mg./kg.l average tration day) degree of Compound in diet X days infection 2-(2-amino-5-thiadiazolyl)-1- methyl-S-nitroimidazole 0. 0125 17 85 (77) 2-(2-acetmnido-5-thiadiazolyl)-1- methyl-5-nitroimidazole 0. 025 32 95 2-(2-methylamino-5-thiadiazoly1)-1- methyl-5-nitroimidazole 0. 0125 17 76 2-[2-(4-carbethoxy-1-piperazinyl)-5- thiadiazolyl]-1-methyl-5-n1troimidazole 0. 05 53 60 2-(2-dimethylamino-5-thiadiazolyl)- l-methyl-nitroimidazole 0. 05 72 74 2-dichloroacetamido5-(1-methy1-5- 5-nitro-2-imidaz0lyl)-1,3,4-thiadiazole 0. 05 55 85 2-[2-(3-dimethylaminopropylamino)-5-thiadiazolyl]-1-methy1- S-nitroimidazole 0. 05 75 66 2-(2-amino-5-oxadiazolyl)-1-methyl- 5-nitroimidazo1e 0. 05 52 62 2-(amino-5-thiadiazolyl)-1-(2-hydroxyethyl)-5-nitroimidazole e. 0. 006 8 03 2-(Z-methylamino-5-oxadiazolyl)-1- methyl-S-nitroimidazole 0. 025 30 57 2-(2-amino-5-thiadiazolyl)-1-ethy1-5- nitroirm'dazole 0.0125 16 62 2-(2-amino-5-oxadiazolyl)-1-ethy1-5- nitroimidazole 0. 025 34 76 2-(2-arnino-5-thiadiazolyl) -1-benzyl- 5-nitroimidazo1e 0. 025 24 83 2-(2-hydroxymethylamino-5-thiadiazolyl)-1-methyl-5-nitr0irnidazole 0. 0125 15 (16) 73 (75) 2-[2-(hydroxyeth ylamino)-5-thiadiazolyl]-l-methyl-5-nitr0imldazole 0. 025 35 88 2-{2-[N-(2-hydroxyethyl)methylamino1-5-thiadiazolyll-5-nitroimidazole 0. 025 32 70 2-[2-(2-morpholinoacetamido)-5- thiadiazolyll-l-methyHi-nitroimldazole 0. 0125 16 71 2-(Z-amino-5-thiadiazoly1)-1-methyl- 5-nitroimidaz0le hydrochloride"-.. 0.0125 17 78 (75) 2-(2-benzylamino-5-thiad1azolyl)- l-methyl-S-nitroimidazole 0.025 26 56 2-(2-ethylamino-5-thiadiaz0lyl)- l-methyl-S-nitroimidazole 0. 0125 22 76 2-(2-cyelohexylamino-5-thiadiazolyl)-1-methyl-5-nitronnidazole 0. 025 30 79 2-(2-t-butylamino-5-thiadiazolyl)- 1-methyl-5-nitroimidazole 0. 025 26 61 2-l2-[4-(3-dirnethylaminopro yl)-1- piperazinyl]-5-thiadiazoly1 -1- methyl-S-nitroimidazole 0. 025 35 100 2-[Z-n-hexylamino-5-thiadiazoly1)-1- methy1-5-nitroimidazole 0. 025 32 71 2-(2-piperidino-5-thiadiazolyl)-1- methyl-5-nitroimidazole 0. 025 32 80 2-l-[4-(2-pyridyl)-1-piperazmyl]-5- thiadiazolyll-l-methyl-5- nitroimidazolo 0. 025 35 58 2-l2-[4'(2-thiazolyl)1-piperazinyl]- S-thiadiazolyl l-l-methyl-S- nitrcimidazole... 0. 025 37 69 2-[2-(1-piporazinyD-5 a l-methyl-S-nitroimidazole 0. 025 24 77 2-[4-(2-hydroxyethy1)-1-piperaz1nyl- 5-thiadiazolyH-Lmethyl-S- nitroimidazole 0. 025 35 65 TABLE VIContinued Approxi Percent Percent mate suppresdosage sion of concen- (mg./kg./ average tratlpn day) degree of Compound in diet X5 days infection 2-{2-[4-(3-dirnethylaminopropyl)- piperidino]-5-thiadiazolyl)-1- methyl-5-nitroimidazole 0. 025 32 60 2-(2m-octylanfino-thiadiazolyl)-1- methyl-S-nitroimidazole 0. 025 34 100 2-[2-(2,2-diethoxyethylamin0)-5- thiadiazolyl]-1-methyl-5- nitroimidazole 0.025 34 89 2-[2-(1-pyrrolidinyl)-5-thiadiazolyl} 1-methyl-5-nitroimidazole 0. 025 32 71 2-[2-(4-hydroxvpipezidino)-5- thladiazolyl1-1-methyl-5- nitroimidazole 0. 025 31 72 2-(2-diethylamino-5-thiadiazolyl)-1- methyl-5-nitroimidazole 0. 025 34 73 2-[2-(Z-dimethylaminoethyl)aminofi-thiadiazolyll-l-methyl-finitroimidazole 0. 025 83 70 2-[2-(1-ethyl-3-piperidylamino)-5- thiadiazolyll-l-rnethyl-5- nitroimidazole 0. 025 31 89 EXAMPLE 59 Testing of mice against the infection Trypanosoma equzperzdum Female albino mice were inoculated intraperitoneally with approximately 100,000 Trypanosoma equiperidum. Three to seven days post-inoculation, untreated animals died. Mice which received active compounds lived longer than the sham-dosed controls or survived until the termination of the assay 14 days post-inoculation. In these experiments, the compounds were administered once, two to four hours post-inoculation. All dosages were based on average mouse weights obtained just before treatment and were corrected for acid or base content. Regimens which results in survival of 50% or more of the mice for 14 days post-inoculation were considered highly suppressive. Mortality of infected untreated controls in this test is typically greater than 99% The following Table VII summarizes the results of the tests:

TABLE VII Number protected/number tested Dose -100 40-50 20-25 1012 Compound:

2-(2-amino-5-thiadiazolyl)-1-methy1- 5-nitroimidazole 2-(2-methylamino-5-thiadiazolyl)-1- methyl-5-nitroimidazo1e 2-[2-(4-carbethoxy-l-plperazinyl)-5- thiadiazolyll-l-methyl-5-nitroimidazole 2-[2-(3-dimethylarninopropylamino)- 5-thiadiazoly11-1-methyl-5-nitromidazole 2-(2-formamido-5-thiadiazolyl)-1- methyl-5-nitroimidazole 2-(2-amino-5-oxadiazolyl)-1-methylfi-nitrclmidazole 5/5 2-(2-dirnethylamino-5-oxadiazolyl)- 1-methyl-5-nitroimidazole 4/5 2-(2-amino-5-thiadiazolyl)-1-(2- hydroxyethyl)-5-nitroirn.idazole 20/20 2-(2-methylamino-5oxadiazolyl)-1- TABLE VII-Continued Number protected/number tested Six female adult patients were treated for ten days with doses of 250 milligrams three times daily of 2-(2- amino-S-thiadiazolyl)-1-methyl 5 nitroimidazole. This amounted to a total daily dose of approximately 12 mg./ kg. The individuals were cultured initially for T. vaginalis infection and cultures were repeated on day 6 and on day 11 and a final culture was obtained three weeks after treatment. Routine blood chemistry was run on each patient before treatment and repeated on day 6 and day 11. There were no signs of toxicity as reflected in the routine blood chemistry. The results obtained are shown in the following Table VIH:

tAll individuals showed positive T. vaginalis injection at beginning of S Presumably due to reinfection.

We claim:

1. A method of inhibiting the growth of protozoa in warm-blooded animals which comprises administering orally or parenterally to said animals an antiprotozoally etfectve amount of a nitroamidazole of the formula:

MINE.-.

i X It 24 wherein R is selected from the group consisting of lower alkyl, hydroxy lower alkyl, lower alkanoyloxy lower alkyl and benzyl; X is selected from the group consisting of oxygen and sulfur; and

taken together is selected from the group consisting of 1- imidazolyl and l-pyrrolidinyl, or physiologically acceptable salts thereof.

2. A therapeutic composition useful for the control of protozoa in warm-blooded animals comprising an edible carrier and an effective protozoal controlling amount of a nitroimidazole of the formula:

wherein R is selected from the group consisting of lower alkyl, hydroxy lower alkyl, lower alkanoyloxy lower alkyl and benzyl; X is selected from the group consisting of oxygen and sulfur; and

taken together is selected from the group consisting of l-imidazolyl and l-pyrrolidinyl, or physiologically acceptable salts thereof.

3. The method of Claim 1 wherein said nitroimidazole is 2-[2-(l-pyrrolidinyl)-5-thiadiazolyl] 1 methyl-5- nitroimidazole.

4. The therapeutic composition of Claim 2 where said nitroimidazole is 2-[2-(l-pyrrolidinyl)-5-thiadiazolyl]-1- methyl-S-nitroimidazole.

References Cited UNITED STATES PATENTS 3,025,303 3/1962 Ifversen et al 260306.8

ALBERT T. MEYERS, Primary Examiner F. E. WADDELL, Assistant Examiner US. Cl. X.R.

I UNITED STATES PATENT OFFICE f l CERTIFICATE OF CORRECTION Patent No 3, 7 v Dated October 5319 Invento.-r(s) GERALD BERKELHAMMER and GORO ASATQ It is certified that error appeers in the above-identified patent and that saidLetters Patent are hereby corrected as shown below:

line 32, "3,666,806" should read 3,666,866 tPaper No. 4, page 2);, i 1

, 001-. 3," line 36, "elkanoy" should read -a'lkanoyl' 1 [Specification page 5, line 17];

061. 4, line 56, "dodecahydrato" should read dodecehydrate [Specification page 8, line 6 ,2];

Col. 5, line'l9, "2l8C ";yshould read 23 ,8'C

[Specification page 9, line 1]; 4 I

Col. 5, line 58, 'ethylthisemic'arbaz'ide" should read ---ethylthiosemicarbazide [Specification page 9, lines, 32-35];

Col. 7, line 17, "635" should read 6.35 [Specification page 12, line 18]; I I

Col. 23, lint-3151 (Claim 1) "nitroamidazole" should read.

' hitroimidazple [Amendment dated May 24, 1973-,

page 1, Claim 1-] ned and sealed; this ch-da of M r- :5 1975, "A

(SEAL) Attest: v

- t r C. MARSHALL DANN I RUTH C. MASON Y Comissioner of Patents Attestins Officer end vT redemetice FORM F'O-IOSO (no-'59) USQOMM-DC 608764 60 i u. s. eovlllnnllv unmet OIIICI I". o-lll-lu 

1. A METHOD OF INHIBITING THE GROWTH OFF PROTOZOA IN WARM-BLOODED ANIMALS WHICH COMPRISES ADMINISTERING ORALLY OR PARENTERALLY TO SAID ANIMALS AN ANTIPROTOZOALLY EFFECTIVE AMOUNT OF A NITROAMIDAZOLE OF THE FORMULA: 